Pectate lyases

ABSTRACT

The present invention relates to microbial pectate lyases, more specifically to microbial enzymes exhibiting pectate lyase activity as their major enzymatic activity in the neutral and alkaline pH ranges, to a method of producing such an enzyme, and to methods for using such enzymes in the textile, detergent and cellulose fiber processing industries.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/073,684filed May 6, 1998 now U.S. Pat. No. 6,124,127 and claims priority under35 U.S.C. 119 of Danish application 1344/97 filed Nov. 24, 1997 and ofU.S. Provisional application No. 60/067,240 filed Dec. 2, 1997, thecontents of which are fully incorporated herein by reference.

The present invention relate to microbial pectate lyase, morespecifically to microbial enzyme exhibiting pectate lyase activity astheir major enzymatic activity in neutral and alkaline pH ranges; to amethod of producing such enzymes; and a method for using such enzymes inthe textile, detergent, and cellulose fiber processing industries.

BACKGROUND OF THE INVENTION

Pectin polymers are important constituents of plant cell walls. Pectinis a hetero-polysaccharide with a backbone composed of alternatinghomogalacturonan (smooth regions) and rhamnogalacturonan (hairyregions). The smooth regions are linear polymers of 1,4-linkedalpha-D-galacturonic acid. The galacturonic acid residues can bemethyl-esterified on the carboxyl group to a varying degree, usually ina non-random fashion with blocks of polygalacturonic acid beingcompletely methyl-esterified.

Pectinases can be classified according to their preferential substrate,highly methyl-esterified pectin or low methyl-esterified pectin andpolygalacturonic acid (pectate), and their reaction mechanism,beta-elimination or hydrolysis. Pectinases can be mainly endo-acting,cutting the polymer at random sites within the chain to give a mixtureof oligomers, or they may be exo-acting, attacking from one end of thepolymer and producing monomers or dimers. Several pectinase activitiesacting on the smooth regions of pectin are included in theclassification of enzymes provided by the Enzyme Nomenclature (1992)such as pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10),polygalacturonase (EC 3.2.1.15), exo-polygalacturonase (EC 3.2.1.67),exo-polygalacturonate lyase (EC 4.2.2.9) andexo-poly-alpha-galacturonosidase (EC 3.2.1.82).

Pectate lyases have been cloned from different bacterial genera such asErwinia, Pseudomonas, Klebsiella and Xanthomonas. Also from Bacillussubtilis (Nasser et al. (1993) FEBS 335:319-326) and Bacillus sp. YA-14(Kim et al. (1994) Biosci. Biotech. Biochem. 58:947-949) cloning of apectate lyase has been described. Purification of pectate lyases withmaximum activity in the pH range of 8-10 produced by Bacillus pumilus(Dave and Vaughn (1971) J. Bacteriol. 108:166-174), B. polymyxa (Nageland Vaughn (1961) Arch. Biochem. Biophys. 93:344-352), B.stearothermophilus (Karbassi and Vaughn (1980) Can. J. Microbiol.26:377-384), Bacillus sp. (Hasegawa and Nagel (1966) J. Food Sci.31:838-845) and Bacillus sp. RK9 (Kelly and Fogarty (1978) Can. J.Microbiol. 24:1164-1172) has been reported, however, no publication wasfound on cloning of pectate lyase encoding genes from these organisms.All the pectate lyases described require divalent cations for maximumactivity, calcium ions being the most stimulatory.

Generally, pectinase producing microorganisms exhibit a broad range ofpectin degrading or modifying enzymes. Often the microorganisms alsoproduce cellulases and/or hemicellulases and complex multi-componentenzyme preparations from such microorganisms may be difficult tooptimise for various applications, they even may contain enzymes withdetrimental effect. Thus, it is an object of the present invention toprovide a pectin degrading enzyme exhibiting only the desired effectse.g. in detergents or different industrial processes.

SUMMARY OF THE INVENTION

The inventors have now found a novel enzyme having substantial pectatelyase activity, i.e. an enzyme exhibiting pectate lyase activity whichmay be obtained from a bacterial strain of the genus Bacillus, morespecifically of the strain Bacillus licheniformis, and have succeeded inidentifying a DNA sequence encoding such enzyme. The DNA sequence andthe deduced amino acid sequence are listed in the sequence listing asSEQ ID No. 1 and 2, respectively. It is believed that the novel enzymewill be classified according to the Enzyme Nomenclature in the EnzymeClass EC 4.2.2.2. However, it should be noted that the enzyme of theinvention also exhibits catalytic activity on pectin (which may beesterified) besides the activity on pectate and polygalacturonidesconventionally attributed to enzymes belonging to EC 4.2.2.2.

In a first aspect, the present invention relates to a pectate lyasewhich is i) a polypeptide produced by Bacillus licheniformis, ATCC14580, or ii) a polypeptide comprising an amino acid sequence as shownin positions 1-341 of SEQ ID NO:2, or iii) an analogue of thepolypeptide defined in i) or ii) which is at least 70% homologous withsaid polypeptide, is derived from said polypeptide by substitution,deletion or addition of one or several amino acids, or isimmunologically reactive with a polyclonal antibody raised against saidpolypeptide in purified form.

Within one aspect, the present invention provides an isolatedpolynucleotide molecule selected from the group consisting of (a)polynucleotide molecules encoding a polypeptide having pectate lyaseactivity and comprising a sequence of nucleotides as shown in SEQ ID NO:1 from nucleotide 1 to nucleotide 1026; (b) species homologs of (a); (c)polynucleotide molecules that encode a polypeptide having pectate lyaseactivity that is at least 70% identical to the amino acid sequence ofSEQ ID NO: 2 from amino acid residue 1 to amino acid residue 341; (d)molecules complementary to (a), (b) or (c); and (e) degeneratenucleotide sequences of (a), (b), (c) or (d).

The plasmid pSJ1678 comprising the polynucleotide molecule (the DNAsequence) encoding a pectate lyase of the present invention has beentransformed into a strain of the Escherichia coli which was deposited bythe inventors according to the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure at the Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Federal Republic ofGermany, on Sep. 25, 1997 under the deposition number DSM 11789.

Within another aspect of the invention there is provided an expressionvector comprising the following operably linked elements: atranscription promoter; a DNA segment selected from the group consistingof (a) polynucleotide molecules encoding a polypeptide having pectatelyase activity and comprising a sequence of nucleotides as shown in SEQID NO: 1 from nucleotide 1 to nucleotide 1026; (b) species homologs of(a); (c) polynucleotide molecules that encode a polypeptide havingpectate lyase activity that is at least 80% identical to the amino acidsequence of SEQ ID NO: 2 from amino acid residue 1 to amino acid residue341; and (d) degenerate nucleotide sequences of (a), (b), or (c); and atranscription terminator.

Within yet another aspect of the present invention there is provided acultured cell into which has been introduced an expression vector asdisclosed above, wherein said cell expresses the polypeptide encoded bythe DNA segment.

A further aspect of the present invention provides an isolatedpolypeptide having pectate lyase activity selected from the groupconsisting of (a) polypeptide molecules comprising a sequence of aminoacid residues as shown in SEQ ID NO:2 from amino acid residue 1 to aminoacid residue 399; (b) species homologs of (a).

Within another aspect of the present invention there is provided acomposition comprising a purified polypeptide according to the inventionin combination with other polypeptides.

Within another aspect of the present invention there are providedmethods for producing a polypeptide according to the inventioncomprising culturing a cell into which has been introduced an expressionvector as disclosed above, whereby said cell expresses a polypeptideencoded by the DNA segment and recovering the polypeptide.

The novel enzyme of the present invention is useful for the treatment ofcellulosic material, especially cellulose-containing fiber, yarn, wovenor non-woven fabric, treatment of mechanical paper-making pulps orrecycled waste paper, and for retting of fibres. The treatment can becarried out during the processing of cellulosic material into a materialready for garment manufacture or fabric manufacture, e.g. in thedesizing or scouring step; or during industrial or household launderingof such fabric or garment.

Accordingly, in further aspects the present invention relates to adetergent composition comprising an enzyme having substantial pectatelyase activity; and to use of the enzyme of the invention for thetreatment of cellulose-containing fibers, yarn, woven or non-wovenfabric.

The enzyme of the invention is very effective for use in an enzymaticscouring process in the preparation of cellulosic material e.g. forproper response in subsequent dyeing operations. Further, it iscontemplated that detergent compositions comprising the novel enzyme arecapable of removing or bleaching certain soils or stains present onlaundry, especially soils and spots resulting from galactan orarabinogalactan containing food, plants, and the like. It is alsocontemplated that treatment with detergent compositions comprising thenovel enzyme can prevent binding of certain soils to the cellulosicmaterial.

Definitions

Prior to discussing this invention in further detail, the followingterms will first be defined.

The term “ortholog” (or “species homolog”) denotes a polypeptide orprotein obtained from one species that has homology to an analogouspolypeptide or protein from a different species.

The term “paralog” denotes a polypeptide or protein obtained from agiven species that has homology to a distinct polypeptide or proteinfrom that same species.

The term “expression vector” denotes a DNA molecule, linear or circular,that comprises a segment encoding a polypeptide of interest operablylinked to additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, and the like.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both. The expression vector of the invention maybe any expression vector that is conveniently subjected to recombinantDNA procedures, and the choice of vector will often depend on the hostcell into which the vector it is to be introduced. Thus, the vector maybe an autonomously replicating vector, i.e. a vector which exists as anextra-chromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

The term “recombinant expressed” or “recombinantly expressed” usedherein in connection with expression of a polypeptide or protein isdefined according to the standard definition in the art. Recombinantlyexpression of a protein is generally performed by using an expressionvector as described immediately above.

The term “isolated”, when applied to a polynucleotide molecule, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985). The term “an isolated polynucleotide” mayalternatively be termed “a cloned polynucleotide”.

When applied to a protein/polypeptide, the term “isolated” indicatesthat the protein is found in a condition other than its nativeenvironment. In a preferred form, the isolated protein is substantiallyfree of other proteins, particularly other homologous proteins (i.e.“homologous impurities” (see below)). It is preferred to provide theprotein in a greater than 40% pure form, more preferably greater than60% pure form.

Even more preferably it is preferred to provide the protein in a highlypurified form, i.e., greater than 80% pure, more preferably greater than95% pure, and even more preferably greater than 99% pure, as determinedby SDS-PAGE.

The term “isolated protein/polypeptide may alternatively be termed“purified protein/polypeptide”.

The term “homologous impurities” means any impurity (e.g. anotherpolypeptide than the polypeptide of the invention) which originate fromthe homologous cell where the polypeptide of the invention is originallyobtained from.

The term “obtained from” as used herein in connection with a specificmicrobial source, means that the polynucleotide and/or polypeptideproduced by the specific source, or by a cell in which a gene from thesource have been inserted.

The term “operably linked”, when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator

The term “polynucleotide” denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules.

The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “promoter” denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5′ non-coding regions of genes.

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “pectin” denotes pectate, polygalacturonic acid, and pectinwhich may be esterified to a higher or lower degree.

The term “pectinase” denotes a pectinase enzyme defined according to theart where pectinases are a group of enzymes that cleave glycosidiclinkages of pectic substances mainly poly(1,4-alpha-D-galacturonide andits derivatives(see reference Sakai et al., Pectin, pectinase andprotopectinase: production, properties and applications, pp 213-294 in:Advances in Applied Microbiology vol:39,1993).

Preferably a pectinase of the invention is a pectinase enzyme whichcatalyzes the random cleavage of alpha-1,4-glycosidic linkages in pecticacid also called polygalacturonic acid by transelimination such as theenzyme class polygalacturonate lyase (EC 4.2.2.2) (PGL) also known aspoly(1,4-alpha-D-galacturonide) lyase also known as pectate lyase.

DETAILED DESCRIPTION OF THE INVENTION

HOW TO USE A SEQUENCE OF THE INVENTION TO GET OTHER RELATED SEQUENCES:The disclosed sequence information herein relating to a polynucleotidesequence encoding a pectate lyase of the invention can be used as a toolto identify other homologous pectate lyases. For instance, polymerasechain reaction (PCR) can be used to amplify sequences encoding otherhomologous pectate lyases from a variety of microbial sources, inparticular of different Bacillus species.

Assay for Activity Test

A polypeptide of the invention having pectate lyase activity may betested for pectate lyase activity according to standard test proceduresknown in the art, such as by applying a solution to be tested to 4 mmdiameter holes punched out in agar plates containing 0.2% AZCL galactan(Megazyme, Australia).

Polynucleotides

Within preferred embodiments of the invention an isolated polynucleotideof the invention will hybridize to similar sized regions of SEQ ID No.1, or a sequence complementary thereto, under at least medium stringencyconditions.

In particular polynucleotides of the invention will hybridize to adenatured double-stranded DNA probe comprising either the full sequenceshown in positions 1-1026 of SEQ ID NO:1 or any probe comprising asubsequence of SEQ ID NO:1 having a length of at least about 100 basepairs under at least medium stringency conditions, but preferably athigh stringency conditions as described in detail below. Suitableexperimental conditions for determining hybridization at medium, or highstringency between a nucleotide probe and a homologous DNA or RNAsequence involves presoaking of the filter containing the DNA fragmentsor RNA to hybridize in 5×SSC (Sodium chloride/Sodium citrate, Sambrooket al. 1989) for 10 min, and prehybridization of the filter in asolution of 5×SSC, 5×Denhardt's solution (Sambrook et al. 1989), 0.5%SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook etal. 1989), followed by hybridization in the same solution containing aconcentration of 10 ng/ml of a random-primed (Feinberg, A. P. andVogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled(specific activity higher than 1×109 cpm/μg ) probe for 12 hours at ca.45° C. The filter is then washed twice for 30 minutes in 2×SSC, 0.5% SDSat least 60° C. (medium stringency), still more preferably at least 65°C. (medium/high stringency), even more preferably at least 70° C. (highstringency), and even more preferably at least 75° C. (very highstringency).

Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using a x-ray film.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the art. DNA and RNA encoding genes of interes can becloned in Gene Banks or DNA libraries by means of methods known in theart.

Polynucleotides encoding polypeptides having pectate lyase activity ofthe invention are then identified and isolated by, for example,hybridization or PCR.

The present invention further provides counterpart polypeptides andpolynucleotides from different bacterial strains (orthologs orparalogs). Of particular interest are pectate lyase polypeptides fromgram-positive alkalophilic strains, including species of Bacillus.

Species homologues of a polypeptides pectate lyase activity of theinvention can be cloned using information and compositions provided bythe present invention in combination with conventional cloningtechniques. For example, DNA can be cloned using chromosomal DNAobtained from a cell type that expresses the protein. Suitable sourcesof DNA can be identified by probing Northern blots with probes designedfrom the sequences disclosed herein. A library is then prepared fromchromosomal DNA of a positive cell line. A DNA encoding an polypeptidehaving pectate lyase activity of the invention can then be isolated by avariety of methods, such as by probing with a complete or partial DNA orwith one or more sets of degenerate probes based on the disclosedsequences. A DNA can also be cloned using the polymerase chain reaction,or PCR (Mullis, U.S. Pat. No. 4,683,202), using primers designed fromthe sequences disclosed herein. Within an additional method, the DNAlibrary can be used to transform or transfect host cells, and expressionof the DNA of interest can be detected with an antibody (mono-clonal orpolyclonal) raised against the pectate lyase cloned from B.licheniformis, ATCC 14580, expressed and purified as described inMaterials and Methods and Example 1, or by an activity test relating toa polypeptide having pectate lyase activity. Similar techniques can alsobe applied to the isolation of genomic clones.

The polypeptide encoding part of the DNA sequence cloned into plasmidpSJ1678 present in Escherichia coli DSM 11789 and/or an analogue DNAsequence of the invention may be cloned from a strain of the bacterialspecies Bacillus licheniformis, preferably the strain ATCC 14580,producing the enzyme with pectin degrading activity, or another orrelated organism as described herein.

Alternatively, the analogous sequence may be constructed on the basis ofthe DNA sequence obtainable from the plasmid present in Escherichia coliDSM 11789, e.g be a sub-sequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the pectat lyase encoded by the DNA sequence, but whichcorresponds to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence (i.e. a variantof the pectin degrading enzyme of the invention).

Polypeptides

The sequence of amino acids no. 1-341 of SEQ ID No 2 is a mature pectatelyase sequence.

The present invention also provides pectate lyase polypeptides that aresubstantially homologous to the polypeptides of SEQ ID NO:2 and theirspecies homologs (paralogs or orthologs. The term “substantiallyhomologous” is used herein to denote polypeptides having 80%, morepreferably at least 85%, and even more preferably at least 90%, sequenceidentity to the sequence shown in SEQ ID NO:2 or their orthologs orparalogs. Such polypeptides will more preferably be at least 95%identical, and most preferably 98% or more identical to the sequenceshown in SEQ ID NO:2 or its orthologs or paralogs. Percent sequenceidentity is determined by conventional methods, by means of computerprograms known in the art such as GAP provided in the GCG programpackage (Program Manual for the Wisconsin Package, Version 8, August1994, Genetics Computer Group, 575 Science Drive, Madison, Wis. USA53711) as disclosed in Needleman, S. B. and Wunsch, C. D., (1970),Journal of Molecular Biology, 48, 443-453, which is hereby incorporatedby reference in its entirety. GAP is used with the following settingsfor polypeptide sequence comparison: GAP creation penalty of 3.0 and GAPextension penalty of 0.1.

Sequence identity of polynucleotide molecules is determined by similarmethods using GAP with the following settings for DNA sequencecomparison: GAP creation penalty of 5.0 and GAP extension penalty of0.3.

The enzyme preparation of the invention is preferably derived from amicroorganism, preferably from a bacterium, an archea or a fungus,especially from a bacterium such as a bacterium belonging to Bacillus,preferably to an alkalophilic Bacillus strain which may be selected fromthe group consisting of the species Bacillus licheniformis and highlyrelated Bacillus species in which all species preferably are at least95%, even more preferably at least 98%, homologous to Bacilluslicheniformis based on aligned 16S rDNA sequences.

Substantially homologous proteins and polypeptides are characterized ashaving one or more amino acid substitutions, deletions or additions.These changes are preferably of a minor nature, that is conservativeamino acid substitutions (see Table 2) and other substitutions that donot significantly affect the folding or activity of the protein orpolypeptide; small deletions, typically of one to about 30 amino acids;and small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20-25 residues, or a small extension that facilitates purification (anaffinity tag), such as a poly-histidine tract, protein A (Nilsson etal., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991.See, in general Ford et al., Protein Expression and Purification 2:95-107, 1991, which is incorporated herein by reference. DNAs encodingaffinity tags are available from commercial suppliers (e.g., PharmaciaBiotech, Piscataway, N.J.; New England Biolabs, Beverly, Mass.).

However, even though the changes described above preferably are of aminor nature, such changes may also be of a larger nature such as fusionof larger polypeptides of up to 300 amino acids or more both as amino-or carboxyl-terminal extensions to a Pectate lyase polypeptide of theinvention.

TABLE 1 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline and a-methyl serine) may be substituted for amino acidresidues of a polypeptide according to the invention. A limited numberof non-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, or preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Essential amino acids in the pectate lyase polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-1085, 1989). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (i.e pectate lyase activity) to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306-312,1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al.,FEBS Lett. 309:59-64, 1992. The identities of essential amino acids canalso be inferred from analysis of homologies with polypeptides which arerelated to a polypeptide according to the invention.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis, recombination and/or shuffling followed by arelevant screening procedure, such as those disclosed by Reidhaar-Olsonand Sauer (Science 241:53-57, 1988), Bowie and Sauer (Proc. Natl. Acad.Sci. USA 86:2152-2156, 1989), WO95/17413, or WO 95/22625. Briefly, theseauthors disclose methods for simultaneously randomizing two or morepositions in a polypeptide, or recombination/shuffling of differentmutations (WO95/17413, WO95/22625), followed by selecting for functionala polypeptide, and then sequencing the mutagenized polypeptides todetermine the spectrum of allowable substitutions at each position.Other methods that can be used include phage display (e.g., Lowman etal., Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Pat. No.5,223,409; Huse, WIPO Publication WO 92/06204) and region-directedmutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA7:127, 1988).

Mutagenesis/shuffling methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that are substantiallyhomologous to residues 1 to 341 of SEQ ID NO: 2 and retain the pectatelyase activity of the wild-type protein.

The pectin degrading enzyme of the invention may, in addition to theenzyme core comprising the catalytically domain, also comprise acellulose binding domain (CBD), the cellulose binding domain and enzymecore (the catalytically active domain) of the enzyme being operablylinked. The cellulose binding domain (CBD) may exist as an integral partof the encoded enzyme, or a CBD from another origin may be introducedinto the pectin degrading enzyme thus creating an enzyme hybrid. In thiscontext, the term “cellulose-binding domain” is intended to beunderstood as defined by Peter Tomme et al. “Cellulose-Binding Domains:Classification and Properties” in “Enzymatic Degradation of InsolubleCarbohydrates”, John N. Saddler and Michael H. Penner (Eds.), ACSSymposium Series, No. 618, 1996. This definition classifies more than120 cellulose-binding domains into 10 families (I-X), and demonstratesthat CBDs are found in various enzymes such as cellulases, xylanases,mannanases, arabinofuranosidases, acetyl esterases and chitinases. CBDshave also been found in algae, e.g. the red alga Porphyra purpurea as anon-hydrolytic polysaccharide-binding protein, see Tomme et al., op.cit. However, most of the CBDs are from cellulases and xylanases, CBDsare found at the N and C termini of proteins or are internal. Enzymehybrids are known in the art, see e.g. WO 90/00609 and WO 95/16782, andmay be prepared by transforming into a host cell a DNA constructcomprising at least a fragment of DNA encoding the cellulose-bindingdomain ligated, with or without a linker, to a DNA sequence encoding thepectin degrading enzyme and growing the host cell to express the fusedgene. Enzyme hybrids may be described by the following formula:

CBD-MR-X

wherein CBD is the N-terminal or the C-terminal region of an amino acidsequence corresponding to at least the cellulose-binding domain; MR isthe middle region (the linker), and may be a bond, or a short linkinggroup preferably of from about 2 to about 100 carbon atoms, morepreferably of from 2 to 40 carbon atoms; or is preferably from about 2to to about 100 amino acids, more preferably of from 2 to 40 aminoacids; and X is an N-terminal or C-terminal region of the pectindegrading enzyme of the invention.

Preferably, the enzyme of the present invention has its maximumcatalytic activity at a pH of at least 8, more preferably of at least8.5, more preferably of at least 9, more preferably of at least 9.5,more preferably of at least 10, even more preferably of at least 10.5,especially of at least 11; and preferably the maximum activity of theenzyme is obtained at a temperature of at least 50° C., more preferablyof at least 55° C.

Protein Production

The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Bacterialcells, particularly cultured cells of gram-positive organisms, arepreferred. Gram-positive cells from the genus of Bacillus are especiallypreferred, such as Bacillus subtilis, Bacillus lentus, Bacillus brevis,Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus thuringiensis, Bacillus agaradherens, or in particularBacillus licheniformis.

Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY,1987; and (Bacillus subtilis and Other Gram-Positive Bacteria,Sonensheim et al., 1993, American Society for Microbiology, WashingtonD.C.), which are incorporated herein by reference.

In general, a DNA sequence encoding a pectate lyase of the presentinvention is operably linked to other genetic elements required for itsexpression, generally including a transcription promoter and terminatorwithin an expression vector. The vector will also commonly contain oneor more selectable markers and one or more origins of replication,although those skilled in the art will recognize that within certainsystems selectable markers may be provided on separate vectors, andreplication of the exogenous DNA may be provided by integration into thehost cell genome. Selection of promoters, terminators, selectablemarkers, vectors and other elements is a matter of routine design withinthe level of ordinary skill in the art. Many such elements are describedin the literature and are available through commercial suppliers.

To direct a polypeptide into the secretory pathway of a host cell, asecretory signal sequence (also known as a leader sequence, preprosequence or pre sequence) is provided in the expression vector. Thesecretory signal sequence may be that of the polypeptide, or may bederived from another secreted protein or synthesized de novo. Numeroussuitable secretory signal sequences are known in the art and referenceis made to (Bacillus subtilis and Other Gram-Positive Bacteria,Sonensheim et al., 1993, American Society for Microbiology, WashingtonD.C.; and Cutting, S. M.(eds.) “Molecular Biological Methods forBacillus”. John Wiley and Sons, 1990) for further description ofsuitable secretory signal sequences especially for secretion in aBacillus host cell. The secretory signal sequence is joined to the DNAsequence in the correct reading frame. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences may be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

Protein Isolation

When the expressed recombinant polypeptide is secreted the polypeptidemay be purified from the growth media. Preferably the expression hostcells are removed from the media before purification of the polypeptide(e.g. by centrifugation).

When the expressed recombinant polypeptide is not secreted from the hostcell, the host cell are preferably disrupted and the polypeptidereleased into an aqueous “extract” which is the first stage of suchpurification techniques. Preferably the expression host cells areremoved from the media before the cell disruption (e.g. bycentrifugation).

The cell disruption may be performed by conventional techniques such asby lysozyme digestion or by forcing the cells through high pressure. See(Robert K. Scobes, Protein Purification, Second edition,Springer-Verlag) for further description of such cell disruptiontechniques.

Whether or not the expressed recombinant polypeptides (or chimericpolypeptides) is secreted or not it can be purified using fractionationand/or conventional purification methods and media.

Ammonium sulfate precipitation and acid or chaotrope extraction may beused for fractionation of samples. Exemplary purification steps mayinclude hydroxyapatite, size exclusion, FPLC and reverse-phase highperformance liquid chromatography. Suitable anion exchange media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred,with DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, N.J.) beingparticularly preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers.

Selection of a particular method is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988.

Polypeptides of the invention or fragments thereof may also be preparedthrough chemical synthesis. Polypeptides of the invention may bemonomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue.

Based on the sequence information disclosed herein a full length DNAsequence encoding a pectinase of the invention and comprising the DNAsequence shown in SEQ ID No 1 may be cloned.

Cloning of is performed by standard procedures known in the art such asby,

preparing a genomic library from a Bacillus strain;

plating such a library on suitable substrate plates;

identifying a clone comprising a polynucleotide sequence of theinvention by standard hybridization techniques using a probe based onSEQ ID No 1; or by

identifying a clone from said Bacillus licheniformis ATCC 14580 or DSM8721 genomic library by an Inverse PCR strategy using primers based onsequence information from SEQ ID No 1. Reference is made to M. J.MCPherson et al. (“PCR A practical approach” Information Press Ltd,Oxford England) for further details relating to Inverse PCR.

Based on the sequence information disclosed herein (SEQ ID No 1, SEQ IDNo 2) is it routine work for a person skilled in the art to isolatehomologous polynucleotide sequences encoding homologous pectinase of theinvention by a similar strategy using genomic libraries from relatedmicrobial organisms, in particular from genomic libraries from otherstrains of the genus Bacillus such as Bacillus subtilis.

Alternatively, the DNA encoding the pectin degrading enzyme of theinvention may, in accordance with well-known procedures, conveniently becloned from a suitable source, such as any of the below mentionedorganisms, by use of synthetic oligonucleotide probes prepared on thebasis of the DNA sequence obtainable from the plasmid present inEscherichia coli DSM 11789.

Accordingly, the polynucleotide molecule of the invention may beisolated from Escherichia coli, DSM 11789, in which the plasmid obtainedby cloning such as described above is deposited. Also, the presentinvention relates to an isolated substantially pure biological cultureof the strain Escherichia coli, DSM 11789.

In the present context, the term “enzyme preparation” is intended tomean either be a conventional enzymatic fermentation product, possiblyisolated and purified, from a single species of a microorganism, suchpreparation usually comprising a number of different enzymaticactivities; or a mixture of monocomponent enzymes, preferably enzymesderived from bacterial or fungal species by using conventionalrecombinant techniques, which enzymes have been fermented and possiblyisolated and purified separately and which may originate from differentspecies, preferably fungal or bacterial species; or the fermentationproduct of a microorganism which acts as a host cell for expression of arecombinant pectate lyase, but which microorganism simultaneouslyproduces other enzymes, e.g. pectin lyases, proteases, or cellulases,being naturally occurring fermentation products of the microorganism,i.e. the enzyme complex conventionally produced by the correspondingnaturally occurring microorganism.

The pectate lyase preparation of the invention may further comprise oneor more enzymes selected from the group consisting of proteases,cellulases (endo-β-1,4-glucanases), β-glucanases(endo-β-1,3(4)-glucanases), lipases, cutinases, peroxidases, laccases,amylases, glucoamylases, pectinases, reductases, oxidases,phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,mannanases, xyloglucanases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases, transglutaminases; or mixturesthereof. In a preferred embodiment, one or more or all enzymes in thepreparation is produced by using recombinant techniques, i.e. theenzyme(s) is/are mono-component enzyme(s) which is/are mixed with theother enzyme(s) to form an enzyme preparation with the desired enzymeblend.

In another aspect, the present invention also relates to a method ofproducing the enzyme preparation of the invention, the method comprisingculturing a microorganism capable of producing the pectate lyase underconditions permitting the production of the enzyme, and recovering theenzyme from the culture. Culturing may be carried out using conventionalfermentation techniques, e.g. culturing in shake flasks or fermentorswith agitation to ensure sufficient aeration on a growth medium inducingproduction of the pectate lyase enzyme. The growth medium may contain aconventional N-source such as peptone, yeast extract or casamino acids,a reduced amount of a conventional C-source such as dextrose or sucrose,and an inducer such as xyloglucan or composit plant substrates such ascereal brans (e.g. wheat bran or rice husk). The recovery may be carriedout using conventional techniques, e.g. separation of bio-mass andsupernatant by centrifugation or filtration, recovery of the supernatantor disruption of cells if the enzyme of interest is intracellular,perhaps followed by further purification as described in EP 0 406 314 orby crystallization as described in WO 97/15660.

Immunological Cross-Reactivity

Polyclonal antibodies to be used in determining immunologicalcross-reactivity may be prepared by use of a purified pectate lyaseenzyme. More specifically, antiserum against the pectate lyase of theinvention may be raised by immunizing rabbits (or other rodents)according to the procedure described by N. Axelsen et al. in: A Manualof Quantitative Immunoelectrophoresis, Blackwell ScientificPublications, 1973, Chapter 23, or A. Johnstone and R. Thorpe,Immunochemistry in Practice, Blackwell Scientific Publications, 1982(more specifically p. 27-31). Purified immunoglobulins may be obtainedfrom the antisera, for example by salt precipitation ((NH₄)₂SO₄),followed by dialysis and ion exchange chromatography, e.g. onDEAE-Sephadex. Immunochemical characterization of proteins may be doneeither by Outcherlony double-diffusion analysis (O. Ouchterlony in:Handbook of Experimental Immunology (D. M. Weir, Ed.), BlackwellScientific Publications, 1967, pp. 655-706), by crossedimmunoelectrophoresis (N. Axelsen et al., supra, Chapter 3 and 4), or byrocket immunoelectrophoresis (N. Axelsen et al., Chapter 2).

Examples of useful bacteria producing the enzyme or the enzymepreparation of the invention are Gram positive bacteria, preferably fromthe Bacillus/Lactobacillus subdivision, preferably a strain from thegenus Bacillus, especially a strain of Bacillus licheniformis. ATCC14580 is the type strain of Bacillus licheniformis.

In yet another aspect, the present invention relates to an isolatedpectate lyase having the properties described above and which is freefrom homologous impurities, and is produced using conventionalrecombinant techniques.

Use in the Detergent Industry

In further aspects, the present invention relates to a detergentcomposition comprising the pectate lyase or pectate lyase preparation ofthe invention, and to a process for machine treatment of fabricscomprising treating fabric during a washing cycle of a machine washingprocess with a washing solution containing the pectate lyase or pectatelyase preparation of the invention.

Typically, the detergent composition of the invention comprisesconventional ingredients such as surfactants (anionic, nonionic,zwitterionic, amphoteric), builders, and other ingredients, e.g. asdescribed in WO 97/01629 which is hereby incorporated by reference inits entirety.

Use in the Textile and Cellulosic Fiber Processing Industries

The pectate lyase of the present invention can be used in. combinationwith other carbohydrate degrading enzymes (for instance arabinanase,xyloglucanase, pectinase) for biopreparation of fibers or for cleaningof fibers in combination with detergents. Cotton fibers consist of aprimary cell wall layer containing pectin and a secondary layercontaining mainly cellulose. Under cotton preparation or cotton refiningpart of the primary cell wall will be removed. The present inventionrelates to either help during cotton refining by removal of the primarycell wall. Or during cleaning of the cotton to remove residual pecticsubstances and prevent graying of the textile.

In the present context, the term “cellulosic material” is intended tomean fibers, sewn and unsewn fabrics, including knits, wovens, denims,yarns, and toweling, made from cotton, cotton blends or natural ormanmade cellulosics (e.g. originating from xylan-containing cellulosefibers such as from wood pulp) or blends thereof. Examples of blends areblends of cotton or rayon/viscose with one or more companion materialsuch as wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers,polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose,ramie, hemp, flax/linen, jute, cellulose acetate fibers, lyocell).

The preparation of the present invention is useful in the cellulosicfiber processing industry for the pretreatment or retting of fibers fromhemp, flax or linen.

The processing of cellulosic material for the textile industry, as forexample cotton fiber, into a material ready for garment manufactureinvolves several steps: spinning of the fiber into a yarn; constructionof woven or knit fabric from the yarn and subsequent preparation, dyeingand finishing operations. Woven goods are constructed by weaving afilling yarn between a series of warp yarns; the yarns could be twodifferent types. Knitted goods are constructed by forming a network ofinterlocking loops from one continuous length of yarn. The cellulosicfibers can also be used for non-woven fabric.

The preparation process prepares the textile for the proper response indyeing operations. The sub-steps involved in preparation are

a. Desizing (for woven goods) using polymeric size like e.g. starch, CMCor PVA is added before weaving in order to increase the warp speed; Thismaterial must be removed before further processing.

b. Scouring, the aim of which is to remove non-cellulosic material fromthe cotton fiber, especially the cuticle (mainly consisting of waxes)and primary cell wall (mainly consisting of pectin, protein andxyloglucan). A proper wax removal is necessary for obtaining a highwettability, being a measure for obtaining a good dyeing. Removal of theprimary cell wall—especially the pectins—improves wax removal andensures a more even dyeing. Further this improves the whiteness in thebleaching process. The main chemical used in scouring is sodiumhydroxide in high concentrations, up to 70 g/kg cotton and at hightemperatures, 80-95° C.; and

c. Bleaching; normally the scouring is followed by a bleach usinghydrogen peroxide as the oxidizing agent in order to obtain either afully bleached (white) fabric or to ensure a clean shade of the dye.

A one step combined scour/bleach process is also used by the industry.Although preparation processes are most commonly employed in the fabricstate; scouring, bleaching and dyeing operations can also be done at thefiber or yarn stage.

The processing regime can be either batch or continuous with the fabricbeing contacted by the liquid processing stream in open width or ropeform. Continuous operations generally use a saturator whereby anapproximate equal weight of chemical bath per weight of fabric isapplied to the fabric, followed by a heated dwell chamber where thechemical reaction takes place. A washing section then prepares thefabric for the next processing step. Batch processing generally takesplace in one processing bath whereby the fabric is contacted withapproximately 8-15 times its weight in chemical bath. After a reactionperiod, the chemicals are drained, fabric rinsed and the next chemicalis applied. Discontinuous pad-batch processing involves a saturatorwhereby an approximate equal weight of chemical bath per weight offabric is applied to the fabric, followed by a dwell period which in thecase of cold pad-batch might be one or more days.

Woven goods are the prevalent form of textile fabric construction. Theweaving process demands a “sizing” of the warp yarn to protect it fromabrasion. Starch, polyvinyl alcohol (PVA), carboxymethyl cellulose,waxes and acrylic binders are examples of typical sizing chemicals usedbecause of availability and cost. The size must be removed after theweaving process as the first step in preparing the woven goods. Thesized fabric in either rope or open width form is brought in contactwith the processing liquid containing the desizing agents. The desizingagent employed depends upon the type of size to be removed. For PVAsizes, hot water or oxidative processes are often used. The most commonsizing agent for cotton fabric is based upon starch. Therefore mostoften, woven cotton fabrics are desized by a combination of hot water,the enzyme α-amylase to hydrolyze the starch and a wetting agent orsurfactant. The cellulosic material is allowed to stand with thedesizing chemicals for a “holding period” sufficiently long toaccomplish the desizing. The holding period is dependent upon the typeof processing regime and the temperature and can vary from 15 minutes to2 hours, or in some cases, several days. Typically, the desizingchemicals are applied in a saturator bath which generally ranges fromabout 15° C. to about 55° C. The fabric is then held in equipment suchas a “J-box” which provides sufficient heat, usually between about 55°C. and about 100° C., to enhance the activity of the desizing agents.The chemicals, including the removed sizing agents, are washed away fromthe fabric after the termination of the holding period.

In order to ensure a high whiteness or a good wettability and resultingdyeability, the size chemicals and other applied chemicals must bethoroughly removed. It is generally believed that an efficient desizingis of crucial importance to the following preparation processes:scouring and bleaching.

The scouring process removes much of the non-cellulosic compoundsnaturally found in cotton. In addition to the natural non-cellulosicimpurities, scouring can remove dirt, soils and residual manufacturingintroduced materials such as spinning, coning or slashing lubricants.The scouring process employs sodium hydroxide or related causticizingagents such as sodium carbonate, potassium hydroxide or mixturesthereof. Generally an alkali stable surfactant is added to the processto enhance solubilization of hydrophobic compounds and/or prevent theirredeposition back on the fabric. The treatment is generally at a hightemperature, 80° C.-100° C., employing strongly alkaline solutions, pH13-14, of the scouring agent. Due to the non-specific nature of chemicalprocesses not only are the impurities but the cellulose itself isattacked, leading to damages in strength or other desirable fabricproperties. The softness of the cellulosic fabric is a function ofresidual natural cotton waxes. The non-specific nature of the hightemperature strongly alkaline scouring process cannot discriminatebetween the desirable natural cotton lubricants and the manufacturingintroduced lubricants. Furthermore, the conventional scouring processcan cause environmental problems due to the highly alkaline effluentfrom these processes. The scouring stage prepares the fabric for theoptimal response in bleaching. An inadequately scoured fabric will needa higher level of bleach chemical in the subsequent bleaching stages.

The bleaching step decolorizes the natural cotton pigments and removesany residual natural woody cotton trash components not completelyremoved during ginning, carding or scouring. The main process in usetoday is an alkaline hydrogen peroxide bleach. In many cases, especiallywhen a very high whiteness is not needed, bleaching can be combined withscouring.

In the examples below it is shown that the scouring step can be carriedout using the pectate lyase or pectate lyase preparation of the presentinvention a temperature of about 50° C.-80° C. and a pH of about 7-11,thus substituting or supplementing the highly causticizing agents. Anoptimized enzymatic process ensures a high pectin removal and fullwettability.

Degradation or Modification of Plant Material

The enzyme or enzyme preparation according to the invention ispreferably used as an agent for degradation or modification of plantcell walls or any pectin-containing material originating from plantcells walls due to the high plant cell wall degrading activity of thepectate lyase of the invention.

The pectate lyase of the present invention may be used alone or togetherwith other enzymes like glucanases, pectinases and/or hemicellulases toimprove the extraction of oil from oil-rich plant material, likesoy-bean oil from soy-beans, olive-oil from olives or rapeseed-oil fromrape-seed or sunflower oil from sunflower.

The pectate lyase of the present invention may be used for separation ofcomponents of plant cell materials. Of particular interest is theseparation of sugar or starch rich plant material into components ofconsiderable commercial interest (like sucrose from sugar beet or starchfrom potato) and components of low interest (like pulp or hullfractions). Also, of particular interest is the separation ofprotein-rich or oil-rich crops into valuable protein and oil andinvaluable hull fractions, The separation process may be performed byuse of methods known in the art.

The pectate lyase of the invention may also be used in the preparationof fruit or vegetable juice in order to increase yield, and in theenzymatic hydrolysis of various plant cell wall-derived materials orwaste materials, e.g. from wine or juice production, or agriculturalresidues such as vegetable hulls, bean hulls, sugar beet pulp, olivepulp, potato pulp, and the like.

The plant material may be degraded in order to improve different kindsof processing, facilitate purification or extraction of other componentthan the galactans like purification of pectins from citrus, improve thefeed value, decrease the water binding capacity, improve thedegradability in waste water plants, improve the conversion of plantmaterial to ensilage, etc.

By means of an enzyme preparation of the invention it is possible toregulate the consistency and appearence of processed fruit orvegetables. The consistency and appearence has been shown to be aproduct of the actual combination of enzymes used for processing, i.e.the specificity of the enzymes with which the pectate lyase of theinvention is combined. Examples include the production of clear juicee.g. from apples, pears or berries; cloud stable juice e.g. from apples,pears, berries, citrus or tomatoes; and purees e.g. from carrots andtomatoes.

The pectate lyase of the invention may be used in modifying theviscosity of plant cell wall derived material. For instance, the pectatelyase may be used to reduce the viscosity of feed which contain galactanand to promote processing of viscous galactan containing material. Theviscosity reduction may be obtained by treating the galactan containingplant material with an enyme preparation of the invention under suitableconditions for full or partial degradation of the galactan containingmaterial

The pectate lyase can be used e.g. in combination with other enzymes forthe removal of pectic substances from plant fibres. This removal isessential e.g. in the production of textile fibres or other cellulosicmaterials. For this purpose plant fibre material is treated with asuitable amount of the pectate lyase of the invention under suitableconditions for obtaining full or partial degradation of pecticsubstances associated with the plant fibre material.

Animal Feed Additive

Pectate lyases of the present invention may be used for modification ofanimal feed and may exert their effect either in vitro (by modifyingcomponents of the feed) or in vivo. the pectate lyase is particularlysuited for addition to animal feed compositions containing high amountsof arabinogalactans or galactans, e.g. feed containing plant materialfrom soy bean, rape seed, lupin etc. When added to the feed the pectatelyase significantly improves the in vivo break-down of plant cell wallmaterial, whereby a better utilization of the plant nutrients by theanimal is achieved. Thereby, the growth rate and/or feed conversionratio (i.e. the weight of ingested feed relative to weight gain) of theanimal is improved. For example the indigestible galactan is degraded bypectate lyase, e.g. in combination with β-galactosidase, to galactose orgalactooligomers which are digestible by the animal and thus contributeto the available energy of the feed. Also, by the degradation ofgalactan the pectate lyase may improve the digestibility and uptake ofnon-carbohydrate feed constituents such as protein, fat and minerals.

For further description reference is made to PCT/DK 96/00443 and aworking example herein.

Wine and Juice Processing

The enzyme or enzyme preparation of the invention may be used forde-pectinization and viscosity reduction in vegetable or fruit juice,especially in apple or pear juice. This may be accomplished by treatingthe fruit or vegetable juice with an enzyme preparation of the inventionin an amount effective for degrading pectin-containing materialcontained in the fruit or vegetable juice.

The enzyme or enzyme preparation may be used in the treatment of mashfrom fruits and vegetables in order to improve the extractability ordegradability of the mash. For instance, the enzyme preparation may beused in the treatment of mash from apples and pears for juiceproduction, and in the mash treatment of grapes for wine production.

Determination of Catalytic Activity of Pectate Lyase

The Viscosity Assay APSU

APSU units: The APSU unit assay is a viscosity measurement using thesubstrate polygalacturonic acid with no added calcium.

The substrate 5% polygalacturonic acid sodium salt (Sigma P-1879) issolubilised in 0.1 M Glycin buffer pH 10. The 4 ml substrate ispreincubated for 5 min at 40° C. The enzyme is added (in a volume of 250μl ) and mixed for 10 sec on a mixer at maximum speed, it is thenincubated for 20 min at 40° C. For a standard curve double determinationof a dilution of enzyme concentration in the range of 5 APSU/ml to above100 APSU/ml with minimum of 4 concentrations between 10 and 60 APSU perml The viscosity is measured using a MIVI 600 from the company Sofraser,45700 Villemandeur, France. The viscosity is measured as mV after 10sec.

For calculation of APSU units a enzyme standard dilution as describedabove was used for obtaining a standard curve. The GrafPad Prismprogram, using a non linear fit with a one phase exponential decay witha plateau, was used for calculations. The plateau plus span is the mVobtained without enzyme. The plateau is the mV of more than 100 APSU andthe half reduction of viscosity in both examples was found to be 12 APSUunits with a standard error of 1.5 APSU.

The Lyase Assay (at 235 nm)

For determination of the β-elimination an assay measuring the increasein absorbance at 235 nm was carried out using the substrate 0.1%polygalacturonic acid sodium salt (Sigma P-1879) solubilised in 0.1 MGlycin buffer pH 10. For calculation of the catalytic rate an increaseof 5.2 Absorbency at 235 units per min corresponds to formation of 1μmol of unsaturated product (Nasuna and Starr (1966) J. Biol. Chem. Vol241 page 5298-5306; and Bartling, Wegener and Olsen (1995) MicrobiologyVol 141 page 873-881).

Steady state condition using a 0.5 ml cuvette with a 1 cm light path ona HP diode array spectrophotometer in a temperature controlled cuvetteholder with continuous measurement of the absorbency at 235 nm. Forsteady state a linear increase for at least 200 sec was used forcalculation of the rate. It was used for converted to formation μmol permin product.

Materials and Methods

Strains

Bacillus licheniformis ATCC 14580.

B.subtilis PL2306. This strain is the B.subtilis DN1885 with disruptedapr and npr genes (Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen,B. R., Sjøholm, C. (1990) Cloning of aldB, which encodesalpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis. J.Bacteriol., 172, 4315-4321) disrupted in the transcriptional unit of theknown Bacillus subtilis cellulase gene, resulting in cellulase negativecells. The disruption was performed essentially as described in ( Eds.A. L. Sonenshein, J. A. Hoch and Richard Losick (1993) Bacillus subtilisand other Gram-Positive Bacteria, American Society for microbiology,p.618).

Competent cells were prepared and transformed as described by Yasbin, R.E., Wilson, G. A. and Young, F. E. (1975) Transformation andtransfection in lysogenic strains of Bacillus subtilis: evidence forselective induction of prophage in competent cells. J. Bacteriol,121:296-304.

Plasmids

pMOL944

This plasmid is a pUB110 derivative essentially containing elementsmaking the plasmid propagatable in Bacillus subtilis, kanamycinresistance gene and having a strong promoter and signal peptide clonedfrom the amyL gene of B.licheniformis ATCC14580. The signal peptidecontains a SacII site making it convenient to clone the DNA encoding themature part of a protein in-fusion with the signal peptide. This resultsin the expression of a Pre-protein which is directed towards theexterior of the cell.

The plasmid was constructed by means of conventional genetic engineeringtechniques which are briefly described in the following.

Construction of pMOL944

The pUB110 plasmid (McKenzie, T. et al., 1986, Plasmid 15:93-103) wasdigested with the unique restriction enzyme NciI. A PCR fragmentamplified from the amyL promoter encoded on the plasmid pDN1981 (P. L.Jørgensen et al.,1990, Gene, 96, p37-41.) was digested with NciI andinserted in the NciI digested pUB110 to give the plasmid pSJ2624. Thetwo PCR primers used have the following sequences:

# LWN5494 5′-GTCGCCGGGGCGGCCGCTATCAATTGGTAACTGTATCTCAGC -3′ (SEQ IDNO:3) # LWN5495 5′-GTCGCCCGGGAGCTCTGATCAGGTACCAAGCTTGTCGACCTGCAGAATGAGGCAGCAAGAAGAT -3′ (SEQ ID NO:4)

The primer #LWN5494 inserts a NotI site in the plasmid.

The plasmid pSJ2624 was then digested with SacI and NotI and a new PCRfragment amplified on amyL promoter encoded on the pDN1981 was digestedwith SacI and NotI and this DNA fragment was inserted in the SacI-NotIdigested pSJ2624 to give the plasmid pSJ2670.

This cloning replaces the first amyL promoter cloning with the samepromoter but in the opposite direction. The two primers used for PCRamplification have the following sequences:

#LWN5938 5′-GTCGGCGGCCGCTGATCACGTACCAAGCTTGTCGACCTGCAGAATGAGGCAGCAAGAAGAT -3′ (SEQ ID NO:5) #LWN59395′-GTCGGAGCTCTATCAATTGGTAACTGTATCTCAGC -3′ (SEQ ID NO:6)

The plasmid pSJ2670 was digested with the restriction enzymes PstI andBclI and a PCR fragment amplified from a cloned DNA sequence encodingthe alkaline amylase SP722 (disclosed in the International PatentApplication published as WO95/26397 which is hereby incorporated byreference in its entirety) was digested with PstI and BclI and insertedto give the plasmid pMOL944. The two primers used for PCR amplificationhave the following sequence:

#LWN7864 5′-AACAGCTGATCACGACTGATCTTTTAGCTTGGCAC-3′ (SEQ ID NO:7)#LWN7901 5′ -AACTGCAGCCGCGGCACATCATAATGGGACAAATGGG -3′ (SEQ ID NO:8)

The primer #LWN7901 inserts a SacII site in the plasmid.

Genomic DNA Preparation

Strain Bacillus licheniformis ATCC 14580 was propagated in liquid medium3 as specified by ATCC (American Type Culture Collection, USA). After 18hours incubation at 37° C. and 300 rpm, the cells were harvested, andgenomic DNA isolated by the method described by Pitcher et al. (Pitcher,D. G., Saunders, N. A., Owen, R. J. (1989). Rapid extraction ofbacterial genomic DNA with guanidium thiocyanate. Lett. Appl.Microbiol., 8, 151-156).

The pectate lyase encoding DNA sequence of the invention was PCRamplified using the PCR primer set consisting of these two oligonucleotides:

Pecl.B.lich.upper.SacII 5′-CTA ACT GCA GCC GCG GCA GCT TCT GCC TTA AACTCG GGC -3′ (SEQ ID NO:9) Pecl.B.lich.lower.NotI 5′-GCG TTG AGA CGCGCG GCC  GCT GAA TGC CCC GGA CGT TTC ACC -3′ (SEQ ID NO:10)

Restriction sites SacII and NotII are underlined.

Chromosomal DNA isolated from B.licheniformis ATCC 14580 as describedabove was used as template in a PCR reaction using Amplitaq DNAPolymerase (Perkin Elmer) according to manufacturers instructions. ThePCR reaction was set up in PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mMKCl, 1.5 mM MgCl₂, 0.01% (w/v) gelatin) containing 200 μM of each dNTP,2.5 units of AmpliTaq polymerase (Perkin-Elmer, Cetus, USA) and 100 pmolof each primer

The PCR reactions was performed using a DNA thermal cycler (Landgraf,Germany). One incubation at 94° C. for 1 min followed by thirty cyclesof PCR performed using a cycle profile of denaturation at 94° C. for 30sec, annealing at 60° C. for 1 min, and extension at 72° C. for 2 min.Five-μl aliquots of the amplification product was analysed byelectrophoresis in 0.7% agarose gels (NuSieve, FMC). The appearance of aDNA fragment size 1.0 kb indicated proper amplification of the genesegment.

Subcloning of PCR Fragment.

Fortyfive-μl aliquots of the PCR products generated as described abovewere purified using QIAquick PCR purification kit (Qiagen, USA)according to the manufacturer's instructions. The purified DNA waseluted in 50 μl of 10 mM Tris-HCl, pH 8.5. 5 μg of pMOL944 andtwentyfive-μl of the purified PCR fragment was digested with SacII andNotI, electrophoresed in 0.8% low gelling temperature agarose (SeaPlaqueGTG, FMC) gels, the relevant fragments were excised from the gels, andpurified using QIAquick Gel extraction Kit (Qiagen, USA) according tothe manufacturer's instructions. The isolated PCR DNA fragment was thenligated to the SacII-NotI digested and purified pMOL944. The ligationwas performed overnight at 16° C. using 0.5 μg of each DNA fragment, 1 Uof T4 DNA ligase and T4 ligase buffer (Boehringer Mannheim, Germany).

The ligation mixture was used to transform competent B.subtilis PL2306.The transformed cells were plated onto LBPG-10 μg/ml of Kanamycinplates. After 18 hours incubation at 37° C. several clones wererestreaked on fresh agar plates and also grown in liquid TY cultureswith 10 μg/ml kanamycin and incubated overnight at 37° C. Next day 1 mlof cells were used to isolate plasmid from the cells using the QiaprepSpin Plasmid Miniprep Kit #27106 according to the manufacturersrecommendations for B.subtilis plasmid preparations. This plasmid DNAwas used as template for DNA sequencing.

One clone containing the pectate lyase gene was kept, this clone wastermed MB541.

The DNA corresponding to the mature part of the pectate lyase wascharacterised by DNA sequencing by primerwalking, using the Taqdeoxy-terminal cycle sequencing kit (Perkin-Elmer, USA), fluorescentlabelled terminators and appropriate oligonucleotides as primers.

Analysis of the sequence data was performed according to Devereux et al.(1984) Nucleic Acids Res. 12, 387-395. The cloned DNA sequence wasexpressed in B.subtilis and the protein that appeared in the supernatantcorresponded to the mature protein represented in SEQ ID NO:2.

Media

TY (as described in Ausubel, F. M. et al. (eds.) “Current protocols inMolecular Biology”. John Wiley and Sons, 1995).

LB agar (as described in Ausubel, F. M. et al. (eds.) “Current protocolsin Molecular Biology”. John Wiley and Sons, 1995).

LBPG is LB agar supplemented with 0.5% Glucose and 0.05 M potassiumphosphate, pH 7.0

BPX media is described in EP 0 506 780 (WO 91/09129).

The following examples illustrate the invention.

EXAMPLE 1

Purification and Characterization of Pectate Lyase from Bacilluslicheniformis

MB541 (see Materials and Methods) was grown in 25×200 ml BPX media with10 μg/ml of Kanamycin in 500 ml two baffled shakeflasks for 5 days at37° C. at 300 rpm, whereby 3500 ml ofculture broth was obtained. The pHwas adjusted to 5.0 using acetic acid and 100 ml of cationic agent(C521) and 200 ml of anionic agent (A130) was added during agitation forflocculation. The flocculated material was separated by centrifugationusing a Sorval RC 3B centrifuge at 10000 rpm for 30 min at 6° C. Theresulting supernatant contained 370 APSU per ml in a total volume of3600 ml.

The supernatant was clarified using Whatman glass filters GF/D and C andfinally concentrated on a filtron with a cut off of 10 kDa. The totalvolume of 2000 ml was adjusted to pH 8.5. 50 gram of DEAE A-50 Sephadex(Pharmacia) was swelled in 2000 ml 50 mM Tris pH 8.5. Excess buffer wasdiscarded and the clear concentrated enzyme solution was mixed with theslurry for 15 min. The enzyme was separated from the ion-exchangematerial by suction on a Buchner funnel. The resulting solution wasconcentrated on a filtron with a cut off of 10 kDa.

The solution (700 ml) was formulated using 350 gram of sorbitol giving aproduct MB 541-2 batch 9751.

For obtaining a highly purified pectate lyase a final step usingS-sepharose cation-exchange chromatography was carried out. 50 ml of thesolution of 950 APSU per ml (see above) was adjusted to pH 5.0 usingacetic acid. It was applied to a 50 ml column containing S-Sepharose(Pharmacia) equilibrated with a buffer of 50 mmol sodium acetate pH 5.0

The pectate lyase bound and was eluted using a gradient of 0.5 M sodiumchloride. The pure enzyme gave a single band in SDS-PAGE of 35 kDa andhas a pI of 9.3. The protein concentration was determined using a molarextinction coefficient of 57750 (based on the amino acid compositiondeducted from the sequence). Using the assay of detection the formationof cleavage by the formation of a double bound which can be measured at235 nm the following data were obtained

1. (conditions: pH 10; glycine buffer; no calcium; polygalacturonic acidSigma P-1879 as substrate): 1 μmol per min per mg.

2. (conditions: pH 10; glycine buffer; no calcium; DE 35 (35% esterifiedpectin) as substrate): 4 μmol per min per mg.

The temperature optimum was found to be 65° C.

Rabbit antiserum was raised against the purified pectate lyase usingstandard conditions (0.1 mg protein per rabbit per immunization).

EXAMPLE 2

Pectate Lyase Treatment of Cellulosic Material

Effect of Temperature on Pectin Removal and Wettability

A 100% cotton woven twill fabric, desized Test Fabric #428U,representing a typical cellulosic material, was treated with an aqueousenzyme solution comprising the B. licheniformis pectate lyase of thisinvention, dosed at 9 APSU/g fabric at pH 9 and at a 15:1 liquor ratio.Treatment time was 2 hours and temperature varied between 35-75° C. Thefabric was rinsed well after the enzyme treatment, dried and then dyedwith Ruthenium Red. The dye uptake was measured spectrophotometricallyand is a measure of the residual pectin on the fiber. The percentage ofresidual pectin was calculated using the dye uptake of the startingmaterial as 100% residual pectin and that of fully chemically scouredand bleached fabric as 0%. Results are shown in Table 1. Further, thewettability (drop test—measuring the time in seconds for a drop of waterto become absorbed by the fabric) was measured and compared to a noenzyme control. Results are shown in Table 2.

TABLE 1 (% residual pectin) Temp., ° C. 35 45 55 65 75 no enzyme 100 10093 90 85 enzyme 52 40 32 28 26 an alkaline scouring leaves typically20-25% residual pectin

TABLE 1 (% residual pectin) Temp., ° C. 35 45 55 65 75 no enzyme 100 10093 90 85 enzyme 52 40 32 28 26 an alkaline scouring leaves typically20-25% residual pectin

The beneficial effect of increasing temperature is clearly seen on bothresponses.

EXAMPLE 3

Pectate Lyase Treatment of Cellulosic Material

Effect of pH on Pectin Removal

A 100% cotton woven twill fabric, desized Test Fabric #428U,representing a typical cellulosic material, was treated with an aqueousenzyme solution comprising the B. licheniformis pectate lyase of theinvention, dosed at 9 APSU/g fabric at a 15:1 liquor ratio. Treatmenttime was 2 hours and the temperature 55° C. pH was varied between 8-11.The fabric was rinsed well after the enzyme treatment, dried and thendyed with Ruthenium Red. The dye uptake is measuredspectrophotometrically and is a measure of the residual pectin on thefiber. The percentage of residual pectin was calculated using the dyeuptake of the starting material as 100% residual pectin and that offully chemically scoured and bleached fabric as 0%. The results areshown in Table 3:

TABLE 3 pH 8 9 10 10.5 11 % residual 35 32 30 48 61 pectin

The pH optimum is found to be at app. 9.5, but a good activity isdemonstrated in a very broad alkaline interval.

EXAMPLE 4

Use of the Enzyme of the Invention in Detergents

The purified enzyme obtained as described in example 1 (batch 9751)showed improved cleaning performance when tested at a level of 1 ppm ina miniwash test using a conventional commercial liquid detergent. Thetest was carried out under conventional North American wash conditions.

LITERATURE

Lever, M. (1972) A new reaction for colormetric determination ofcarbohydrates. Anal. Biochem. 47, 273-279.

N. C. Carpita and D. M. Gibeaut (1993) The Plant Journal 3:1-30.

Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B. R., Sjøholm, C.(1990) Cloning of aldB, which encodes alpha-acetolactate decarboxylase,an exoenzyme from Bacillus brevis. J. Bacteriol. 172:4315-4321.

10 1026 base pairs nucleic acid single linear not provided 1 ATGAAGAAATTAATCAGCAT CATCTTTATC TTTGTATTAG GGGTTGTCGG GTCATTGACA 60 GCGGCGGTTTCGGCAGAAGC AGCTTCTGCC TTAAACTCGG GCAAAGTAAA TCCGCTTGCC 120 GACTTCAGCTTAAAAGGCTT TGCCGCACTA AACGGCGGAA CAACGGGCGG AGAAGGCGGT 180 CAGACGGTAACCGTAACAAC GGGAGATCAG CTGATTGCGG CATTAAAAAA TAAGAATGCA 240 AATACGCCTTTAAAAATTTA TGTCAACGGC ACCATTACAA CATCAAATAC ATCCGCATCA 300 AAGATTGACGTCAAAGACGT GTCAAACGTA TCGATTGTCG GATCAGGGAC CAAAGGGGAA 360 CTCAAAGGGATCGGCATCAA AATATGGCGG GCCAACAACA TCATCATCCG CAACTTGAAA 420 ATTCACGAGGTCGCCTCAGG CGATAAAGAC GCGATCGGCA TTGAAGGCCC TTCTAAAAAC 480 ATTTGGGTTGATCATAATGA GCTTTACCAC AGCCTGAACG TTGACAAAGA TTACTATGAC 540 GGATTATTTGACGTCAAAAG AGATGCGGAA TATATTACAT TCTCTTGGAA CTATGTGCAC 600 GATGGATGGAAATCAATGCT GATGGGTTCA TCGGACAGCG ATAATTACAA CAGGACGATT 660 ACATTCCATCATAACTGGTT TGAGAATCTG AATTCGCGTG TGCCGTCATT CCGTTTCGGA 720 GAAGGCCATATTTACAACAA CTATTTCAAT AAAATCATCG ACAGCGGAAT TAATTCGAGG 780 ATGGGCGCGCGCATCAGAAT TGAGAACAAC CTCTTTGAAA ACGCCAAAGA TCCGATTGTC 840 TCTTGGTACAGCAGTTCACC GGGCTATTGG CATGTATCCA ACAACAAATT TGTAAACTCT 900 AGGGGCAGTATGCCGACTAC CTCTACTACA ACCTATAATC CGCCATACAG CTACTCACTC 960 GACAATGTCGACAATGTAAA ATCAATCGTC AAGCAAAATG CCGGAGTCGG CAAAATCAAT 1020 CCATAA 1026341 amino acids amino acid single linear not provided 2 Met Lys Lys LeuIle Ser Ile Ile Phe Ile Phe Val Leu Gly Val Val 1 5 10 15 Gly Ser LeuThr Ala Ala Val Ser Ala Glu Ala Ala Ser Ala Leu Asn 20 25 30 Ser Gly LysVal Asn Pro Leu Ala Asp Phe Ser Leu Lys Gly Phe Ala 35 40 45 Ala Leu AsnGly Gly Thr Thr Gly Gly Glu Gly Gly Gln Thr Val Thr 50 55 60 Val Thr ThrGly Asp Gln Leu Ile Ala Ala Leu Lys Asn Lys Asn Ala 65 70 75 80 Asn ThrPro Leu Lys Ile Tyr Val Asn Gly Thr Ile Thr Thr Ser Asn 85 90 95 Thr SerAla Ser Lys Ile Asp Val Lys Asp Val Ser Asn Val Ser Ile 100 105 110 ValGly Ser Gly Thr Lys Gly Glu Leu Lys Gly Ile Gly Ile Lys Ile 115 120 125Trp Arg Ala Asn Asn Ile Ile Ile Arg Asn Leu Lys Ile His Glu Val 130 135140 Ala Ser Gly Asp Lys Asp Ala Ile Gly Ile Glu Gly Pro Ser Lys Asn 145150 155 160 Ile Trp Val Asp His Asn Glu Leu Tyr His Ser Leu Asn Val AspLys 165 170 175 Asp Tyr Tyr Asp Gly Leu Phe Asp Val Lys Arg Asp Ala GluTyr Ile 180 185 190 Thr Phe Ser Trp Asn Tyr Val His Asp Gly Trp Lys SerMet Leu Met 195 200 205 Gly Ser Ser Asp Ser Asp Asn Tyr Asn Arg Thr IleThr Phe His His 210 215 220 Asn Trp Phe Glu Asn Leu Asn Ser Arg Val ProSer Phe Arg Phe Gly 225 230 235 240 Glu Gly His Ile Tyr Asn Asn Tyr PheAsn Lys Ile Ile Asp Ser Gly 245 250 255 Ile Asn Ser Arg Met Gly Ala ArgIle Arg Ile Glu Asn Asn Leu Phe 260 265 270 Glu Asn Ala Lys Asp Pro IleVal Ser Trp Tyr Ser Ser Ser Pro Gly 275 280 285 Tyr Trp His Val Ser AsnAsn Lys Phe Val Asn Ser Arg Gly Ser Met 290 295 300 Pro Thr Thr Ser ThrThr Thr Tyr Asn Pro Pro Tyr Ser Tyr Ser Leu 305 310 315 320 Asp Asn ValAsp Asn Val Lys Ser Ile Val Lys Gln Asn Ala Gly Val 325 330 335 Gly LysIle Asn Pro 340 42 base pairs nucleic acid single linear not provided 3GTCGCCGGGG CGGCCGCTAT CAATTGGTAA CTGTATCTCA GC 42 64 base pairs nucleicacid single linear not provided 4 GTCGCCCGGG AGCTCTGATC AGGTACCAAGCTTGTCGACC TGCAGAATGA GGCAGCAAGA 60 AGAT 64 61 base pairs nucleic acidsingle linear not provided 5 GTCGGCGGCC GCTGATCACG TACCAAGCTT GTCGACCTGCAGAATGAGGC AGCAAGAAGA 60 T 61 35 base pairs nucleic acid single linearnot provided 6 GTCGGAGCTC TATCAATTGG TAACTGTATC TCAGC 35 35 base pairsnucleic acid single linear not provided 7 AACAGCTGAT CACGACTGATCTTTTAGCTT GGCAC 35 37 base pairs nucleic acid single linear notprovided 8 AACTGCAGCC GCGGCACATC ATAATGGGAC AAATGGG 37 39 base pairsnucleic acid single linear not provided 9 CTAACTGCAG CCGCGGCAGCTTCTGCCTTA AACTCGGGC 39 42 base pairs nucleic acid single linear cDNAnot provided 10 GCGTTGAGAC GCGCGGCCGC TGAATGCCCC GGACGTTTCA CC 42

What is claimed is:
 1. A method for improving the properties ofcellulosic material, said method comprising treating said material withan effective amount of a polypeptide having pectate lyase activity,wherein said polypeptide is selected from the group consisting of: a) apolypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2from residue 1 to residue 341; b) a polypeptide having at least 95%identity to the sequence of SEQ ID NO: 2 from amino acid residue 1 toamino acid residue 341, wherein said identity is determined by the GAPprogram, using a GAP creation penalty of 3.0 and a GAP extension penaltyof 0.1; and c) a polypeptide encoded by a DNA sequence that hybridizesto the DNA sequence of SEQ ID NO:1 under high stringency conditions,wherein said high stringency conditions comprise hybridization in 5×SSCat 45° C. and washing in 2×SSC at 70° C.
 2. The method according toclaim 1, wherein treasting step comprises scouring of said cellulosicmaterial.
 3. The method according to claim 1, wherein the cellulosicmaterial is selected from the group consisting of fibres, yarn, wovenfabric, and non-woven fabric.
 4. A method for degradation ormodification of plant material said method comprising treating the plantmaterial with an effective amount of a polypeptide having pectate lyaseactivity, wherein said polypeptide is selected from the group consistingof: a) a polypeptide comprising an amino acid sequence as shown in SEQID NO: 2 from residue 1 to residue 341; b) a polypeptide having at least95% identity to the sequence of SEQ ID NO: 2 from amino acid residue 1to amino acid residue 341, wherein said identity is determined by theGAP program, using a GAP creation penalty of 3.0 and a GAP extensionpenalty of 0.1; and c) a polypeptide encoded by a DNA sequence thathybridizes to the DNA sequence of SEQ ID NO:1 under high stringencyconditions, wherein said high stringency conditions comprisehybridization in 5×SSC at 45° C. and washing in 2×SSC at 70° C.
 5. Themethod according to claim 4 wherein the plant material is recycled wastepaper, mechanical paper-making pulps or fibres subjected to a rettingprocess.